1. Field of the Invention
A method is described for making a replication-defective HIV virus particle. The present invention is also directed to a HIV virus particle produced according to the method and to a vaccine including the particle.
2. Description of the Related Art
A variety of strategies have evolved in the pursuit of an effective HIV vaccine. One strategy is the use of attenuated viruses. Recent advances in SIV vaccine studies indicate that live attenuated vaccines provide the best protection against challenge with the pathogenic strain of SIV in vaccinated animals (Clements J. E. et al., Journal of Virology 69 (1995): 2737-2744; Daniel, M. D. et al., Science 258 (1992): 1938-1941; Stahl-Hennig C. et al., Journal of General Virology 77 (1996): 2969-2981; Norley, S. et al., Journal of Virology 219 (1996): 195-205; Almond, N. et al., The Lancet 345 (1995): 1342-1344; Wyand, M. S. et al., Journal of Virology 70 (1996): 3724-3733). This animal model suggests that the protective immunity can be elicited by vaccination with attenuated viruses. However, there is a potential risk in using an attenuated virus as a vaccine in humans. This concern is strengthened by studies in which attenuated viruses are shown to cause disease in neonatal rhesus monkeys (Wyand, M. S. et al., Nature Medicine 3 (1997): 32-36; Baba, T. W. et al., Science 267 (1995): 1820-1825) and, further, by the high rate of reversion of some attenuated SIV strains inoculated in monkeys (Cohen, J., Science 278 (1997): 24-25).
Humoral and cellular immune responses can be elicited in rhesus monkeys inoculated with a naked DNA vaccine. However, there is little or no protective immunity observed in vaccinated animals after challenge with SIV (Lu, S. et al., Journal of Virology 70 (1996): 3978-3991). The mechanisms of protective immunity elicited by attenuated live vaccines in vaccinated animals have not been identified. Protective immunity may be due to the continuous expression of viral proteins from the persisting viral genome in the vaccinated monkeys, to the expression of all (or part of) the SIV proteins from endogenous pathways, or to other aspects (e.g., viral replication site) of the immune response to the live, attenuated virus.
U.S. Pat. No. 5,571,712, to Haynes et al. discloses replication-defective HIV virus particles which do not include retroviral RNA. These retroviral particles are acceptable for safety reasons due to their inability to replicate. However, these particles differ from vaccines capable of replication in their lesser ability to elicit protective immunity. Thus, it is expected that larger numbers of particles must be administered to a subject in a course of vaccination. Further, lack of expression of viral proteins in a vaccinated subject will affect the quality of the protective immunity.
Other vaccines are currently under development, which include peptide vaccines, DNA vaccines, multi-valent virus (i.e., recombinant canarypox vaccines) and combination vaccines (i.e., a DNA vaccine combined with a fowlpox booster vaccine) with varying results. (Gold, D., IAVI Report 3 (1998): 1+.)
The efficacy of HIV vaccines is, generally, difficult to evaluate due to the lack of readily accessible animal models (Girard, M. et al., Virology 232 (1997): 98-104). Therefore, it is most desirable to have a replication-defective virus particle with broad tropism, both with regard to range of species it will infect and with regard to the cell types it will infect in a given subject. It is also highly desirable to have a replication-defective virus particle which can produce antigenic HIV proteins in levels and for a duration suitable to elicit a protective immune response, with both cellular and humoral components. These goals have been achieved in the present invention, a replication-defective HIV vaccine pseudotyped with vesicular stomatitis virus G (VSV-G) protein.
A method is provided for producing a replication-defective retrovirus particle, including the steps of providing a DNA molecule which includes a complete retroviral genome; modifying a portion of the pol gene of the DNA molecule including the protease and reverse transcriptase activity coding regions to the extent that the remaining pol gene cannot produce a protease and reverse transcriptase capable of functioning in replication of the genome, thereby forming a polxe2x88x92 construct; transferring the polxe2x88x92 construct into a suitable host cell; prior to, during or after the step of transferring the polxe2x88x92 construct into the host cell, transferring into the host cell a pseudotyping construct and a packaging construct; growing the host cell under conditions suitable for expression of the constructs and for production of replication-defective retrovirus particles; and collecting the virus particles.
The retroviral genome is preferably an HIV virus genome and most preferably an HIV-1 genome. In one embodiment, the polxe2x88x92 construct is plasmid pHXB2 with nucleotides 2621-4552 deleted. The method may further include the step of modifying the retroviral genome to represent protein sequence variations in immunologically variant retroviruses.
The pseudotyping construct is preferably a construct for expressing the vesicular stomatitis virus G protein. The polxe2x88x92 construct, the packaging construct and the pseudotyping construct may be co-transfected or, alternately, at least one of the pseudotyping construct and the packaging construct are carried stably in the genome of the host cell.
The present invention further includes a replication-defective retrovirus particle, including a retroviral genome in which a portion of the pol gene including the protease and reverse transcriptase activity coding regions is modified to the extent that the remaining pol gene cannot produce a protease and reverse transcriptase capable of functioning in replication of the genome. As described above in connection with the method embodiment, the retroviral genome can be an HIV genome and, preferably, an HIV-1 genome. In one embodiment, the retroviral genome is a polxe2x88x92 construct that is derived from plasmid pHXB2 with nucleotides 2621-4552 thereof deleted. The pseudotyping protein may be the vesicular stomatitis virus G protein.
The present invention further includes a vaccine including the above-described virus particle in its variously described embodiment in combination with a pharmaceutically and/or veterinarilly suitable excipient. The excipient can include mono-, di-, oligo- and polysaccharides, viscosity enhancing and/or tackifying agents, polymers, block polymers and cross-linked polymers. The vaccine can be lyophilized for use in reconstituted form or in immunization by scarification. Further, the vaccine can be formulated into a form selected from the group consisting of an oral liquid, an oral capsule, a liquid for parenteral injection, a transdermal or transmucosal device and a suppository and includes a suitable excipient for preparation of the selected form.